Methods for detection and quantification of nucleic acid or protein targets in a sample

ABSTRACT

The invention provides an assay method for detection and/or quantification of a plurality of nucleic acid or protein targets in a sample. In the method probes are used to associate a detectable tag sequence with each of the selected targets present in the sample. Probes or primers sufficient to identify at least 25, and preferably at least 500, different targets are used. The method involves segregating aliquots of the sample from each other and detecting the tag sequences in each aliquot.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/739,660 (filed Apr. 24, 2007), which claims the benefit of priorityto U.S. provisional application No. 60/794,812 (filed Apr. 24, 2006),the entire content of each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention is high-throughput analysis of analytes in asample using a microfluidic device.

BACKGROUND OF THE INVENTION

The ability to detect specific nucleic acid sequences in a sample hasresulted in new approaches in diagnostic and predictive medicine,environmental, food and agricultural monitoring, molecular biologyresearch, and many other fields. High-throughput detection of specifiedepitopes, proteins, and protein complexes promises to have a similarimpact.

Various methods for such analysis have been developed, includingmultiplexing methods that allow simultaneous detection of large numbersof targets. Additional methods, especially methods that allow detectionof many targets across a broad range of concentrations in a sample wouldbe of great benefit.

SUMMARY OF THE INVENTION

In one aspect the invention provides an assay method for detection of aplurality of nucleic acid or protein targets in a sample. In one aspectthe method involves (1) combining target-specific probes with the sampleunder conditions in which the probe binds a target, if the target ispresent in the sample, and where: (i) target-specific probes sufficientto identify at least 25 different targets are used, (ii) each probecomprises a nucleic acid tag sequence, and (iii) the target-specificprobe forms a target-specific probe product associated with the targetif the target is present in the sample, thereby associating the tag withthe target; (2) distributing aliquots of the sample to separatecompartments of a microfluidic device and segregating the aliquots fromeach other; (3) querying each aliquot of the sample for the presence ofa different target-specific probe product, wherein said queryingcomprises detecting the nucleic acid tag sequence if present in thealiquot. In one embodiment target-specific probes sufficient to identifyat least 500 different targets are combined with the sample. In oneembodiment the assay has a dynamic range of at least 4 orders ofmagnitude.

In one embodiment the querying step involves combining each aliquot witha composition containing one or two amplification (e.g., PCR) primersand/or a tag detecting probe. In one embodiment the tag sequence isamplified using PCR. In one embodiment the amplified tag sequence isdetected using a fluorogenic nuclease assay. In one embodiment thetag-detecting probe is a dual-labeled fluorogenic oligonucleotide probe.In one embodiment each aliquot is queried using the same tag-detectingprobe and a different PCR primer pair. In another embodiment eachaliquot is queried using the same PCR primer pair and a differenttag-detecting probe.

In one embodiment the querying step includes quantifying the amount ofthe nucleic acid tag sequence in each aliquot and correlating the amountof the tag sequence with the amount of target in the sample. Forexample, in one embodiment each nucleic acid tag sequence molecule maycorrespond to one target-specific probe, and each target specific-probemay correspond to one molecule of target. In one embodiment theamplified tag sequence is quantified by the continuous measurement ofPCR product accumulation using a dual-labeled fluorogenicoligonucleotide (TaqMan®) probe.

In one embodiment of the invention the target is a nucleic acid moleculeand the target-specific probe is a padlock probe. In another embodimentof the invention the target is a polypeptide (e.g., protein) moleculeand the target-specific probe is a proximity probe.

In one embodiment the microfluidic device is fabricated, at least inpart, from an elastomeric material. In some embodiments, and withoutlimitation, the aliquot has a volume of from 1 picoliter to 500nanoliters, more often from 100 picoliters to 20 nanoliters, even moreoften from 1 nanoliter to 20 nanoliters, and most often from 5nanoliters to 15 nanoliters.

In one embodiment the invention provides an assay method for detectionof a plurality of DNA targets in a sample involving (1) combiningtarget-specific padlock probes sufficient to identify at least 25different targets with the sample under conditions in which the probesbind and cantenate to the target molecules, if present, forming atarget-specific probe product, where each probe comprises a nucleic acidtag sequence and the padlock probes form target-specific probe productsassociated with the target if the target is present in the sample,thereby associating the tag sequence with the target; (2) distributingaliquots of the sample to separate compartments of a microfluidic deviceand segregating the aliquots from each other; and (3) querying eachaliquot for the presence of a different probe molecule catenated to thetarget, where the querying includes detecting, and optionallyquantifying, the tag sequence if present in the aliquot, and wheredetecting and/or quantifying the tag includes amplifying the tagsequence using PCR primers and detecting the amplification using adual-labeled fluorogenic oligonucleotide probe. The presence of amountof tag sequence can be correlated with the presence or amount of targetin the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram of the 48.48 Dynamic Array Microfluidic Device(Fluidigm Corp.) with an integrated input frame. The parallel linesleading from the sample and assay wells are channels for routing fluidsinto the integrated fluid circuit (IFC). The accumulators hold pressurethat operates valves, within the IFC, necessary for routing fluidsduring loading and containing reaction components once the chip has beenloaded.

FIGS. 2A and 2B illustrate methods of the invention showing thatselectivity can be based on either tags or primers. Ellipses indicatecircularized padlock probes that would be formed if a target targetnucleic acid is present. In this illustration there are 48 potential PLPproducts that could be detected in each sample, each with differenttermini and tag sequence (each numbered 1-48). In FIG. 2A selectivity isbased on use of multiple tags, as described in Example 1. Each PLPproduct has a different tag and binds to a different target sequence,and all tags can be amplified using the same forward and reverse primersrecognizing the same forward (F) and reverse (R) priming sites. “}x{”indicates that in each aliquot of sample tags are amplified usinguniversal primers and the resulting amplified tags are detected with atag-specific TaqMan® probe (or other tag-specific tag detecting probe).In FIG. 2B selectivity is based on the use of specific primer pairs, asdescribed in Example 4. Each PLP product has the same tag sequence (T)but different primer binding sites, so that the tags for each productare amplified using different primer pairs (e.g., FP₁+RP₁), where eachprimer pair recognizes a unique combination of forward (F) and reverse(R) priming sites, and each amplified tag can be detected using the same(“universal”) TaqMan® probe (or other tag detecting probe). Analogousprocesses may be carried out using proximity probes to detect proteins.

FIG. 3 illustrates methods of the invention showing an assay of 2304different targets in a single sample, as illustrated in Example 5.Symbols are as in FIG. 2B. The upper portion of the figure illustratespotential products in a 48×48 array (in this illustration 9 products areshown with others indicated by ellipses).

DETAILED DESCRIPTION OF THE INVENTION

1. Matrix-Type Microfluidic Device

The present method is conducted using a matrix-type microfluidic device.A matrix-type device is one that allows the simultaneous pair-wisecombination of a plurality of substrate/reagent pairs in separateisolated reaction chambers, for example, the simultaneous pair-wisecombination of a plurality of targets and probes. Preferably the deviceis configured to contain a different combination of probes and targetsin each of the different chambers. The number of separate reactionchambers is greater than 50, usually greater than 100, more oftengreater than 500, even more often greater than 1000, and sometimesgreater than 5000, or greater than 10,000.

In an aspect of the invention, the matrix-type microfluidic device is aDynamic Array microfluidic device (“DA”). A DA microfluidic device is anmatrix-type microfluidic device, designed to isolate pair-wisecombinations of targets and reagents (e.g., amplification primers,detection probes, PCR primers, TaqMan® probes, etc.), and suited forcarrying out qualitative and quantitative PCR reactions includingreal-time quantitative PCR analysis. In some embodiments, the DAmicrofluidic device is fabricated, at least in part, from an elastomer.Dynamic Arrays are described in PCT publication WO05107938A2 (ThermalReaction Device and Method For Using The Same) and US Pat. PublicationUS20050252773A1, both incorporated by reference. FIG. 21 of thispublication depicts an exemplary matrix design. DAs may incorporatehigh-density matrix designs that utilize fluid communication viasbetween layers of the microfluidic device to weave control lines andfluid lines through the device and between layers, as shown in FIG. 21.By having a fluid lines in multiple layers of an elastomeric block,higher density reaction cell arrangements are possible. AlternativelyDAs may be designed so that all of the reagent and sample channels arein the same elastomeric layer, with control channels in a differentlayer.

FIG. 21 of WO05107938A2 depicts an exemplary matrix design having afirst elastomeric layer 2110 (1st layer) and a second elastomeric layer2120 (2d layer) each having fluid channels formed therein. For example,a reagent fluid channel in the first layer 2110 is connected to areagent fluid channel in the second layer 2120 through a via 2130, whilethe second layer 2120 also has sample channels therein, the samplechannels and the reagent channels terminating in sample and reagentchambers 2180, respectively. The sample and reagent chambers 2180 are influid communication with each other through an interface channel 2150that has an interface valve 2140 associated therewith to control fluidcommunication between each of the chambers 2180 of a reaction cell 2160.In use, the interface is first closed, then reagent is introduced intothe reagent channel from the reagent inlet and sample is introduced intothe sample channel through the sample inlet, containment valves 2170 arethen closed to isolate each reaction cell 2160 from other reaction cells2160. Once the reaction cells 2160 are isolated, the interface valve2140 is opened to cause the sample chamber and the reagent chamber to bein fluid communication with each other so that a desired reaction maytake place. It will be apparent from this (and the description inWO05107938A2) that the DA may be used for reacting M number of differentsamples with N number of different reagents.

Although the Dynamic Array described above in WO05107938 are well suitedfor conducting the assays of the invention, the invention is not limitedto any particular device or design. Any device that partitions a sample,and allows independent pair-wise combinations of reagents and sample maybe used. FIG. 1 is a diagram illustrating the 48.48 Dynamic Array, acommercially available device available from Fluidigm Corp. (South SanFrancisco Calif.). This device is also described infra in Example 6.

In one aspect the invention relates to the use of a partitioningmicrofluidic device for detection and/or quantification of a pluralityof targets in a sample, each target being associated with an amplifiabletag sequence. The number of targets detected is at least 10, more oftenat least 25, still more often at least 50, even more often at least 100,and in some cases 500 or more.

2. Padlock Probes

Padlock probes (PLPs) are long (e.g., about 100 bases) linearoligonucleotides. The sequences at the 3′ and 5′ ends of the probe arecomplementary to adjacent sequences in the target nucleic acid. In thecentral, noncomplementary region of the PLP there is a “nucleic acidtag” sequence that can be used to identify the specific PLP. The tagsequence is flanked by universal priming sites, which allow PCRamplification of the tag. Upon hybridization to the target, the two endsof the PLP oligonucleotide are brought into close proximity and can bejoined by enzymatic ligation. The resulting product is a circular probemolecule catenated to the target DNA strand. Any unligated probes (i.e.,probes that did not hybridize to a target) are removed by addition of anexonuclease. Hybridization and ligation of a PLP requires that both endsegments recognize the target sequence. In this manner, PLPs provideextremely specific target recognition.

Using universal primers, the tag regions of circularized PLPs can beamplified and resulting amplicons detected. For example, TaqMan® realtime PCR can be carried out to detect and quantify the amplicon. Thepresence and amount of amplicon can be correlated with the presence andquantity of target sequence in the sample. For descriptions of PLPs see,e.g., Landegren et al., 2003, Padlock and proximity probes for in situand array-based analyses: tools for the post-genomic era, Comparativeand Functional Genomics 4:525-30; Nilsson et al., 2006, Analyzing genesusing closing and replicating circles Trends Biotechnol. 24:83-8;Nilsson et al., 1994, Padlock probes: circularizing oligonucleotides forlocalized DNA detection, Science 265:2085-8. Each of the thesereferences is incorporated herein by reference.

In an alternative embodiment, the sequences at the 3′ and 5′ ends ofeach padlock probe are complementary to adjacent sequences in the targetnucleic acid, there is a common “tag” sequence that can be used todetect PLP products, and the tag sequence for each probe is flanked by aunique pair of priming sites, which allow PCR amplification of the tag.Upon hybridization to the target, the two ends of the PLPoligonucleotide are brought into close proximity and can be joined byenzymatic ligation. The resulting product is a circular probe moleculecatenated to the target DNA strand. Any unligated probes (i.e., probesthat did not hybridize to a target) are removed by addition of anexonuclease. Specific primers can be used to amplify the tag regions ofcircularized PLPs, and the resulting amplicons detected.

PLP probes may be multiplexed and a large number of different targets(>1000) can be detected in a single reaction by using an equivalentnumber of different PLPs. However, quantitative analysis is problematic.TaqMan® and other FRET probe-based assays are not readily multiplexed,in part because multiplex fluorescence detection using many differentprobes is limited by high fluorescence background when many differentprobes are used in an assay. For this reason, current approaches tomultiplex PLP-based analysis rely on analysis by hybridization ofamplicons to microarrays. However, the dynamic range of array analysisis limited. For example, in a highly optimized system in which arelatively few number of targets were probed, investigators reported adynamic range of only 100-1000. Szemes et al., 2005, Diagnosticapplication of padlock probes—multiplex detection of plant pathogensusing universal microarrays, Nucleic Acids Res. 33(8):e70, incorporatedherein by reference. In contrast, TaqMan®-based approaches generallyhave a dynamic range of 5-8 orders of magnitude or more. Dynamic range,as used here, has its usual meaning in the art and refers to the rangeof concentrations of an analyte that can be accurately measured by anassay. For example, an assay that can accurately measure quantities of 5nanograms and 5 micrograms has a dynamic range of 1000 or 10³. An assaythat can accurately measure quantities of 10 picograms and 10 microgramshas a dynamic range of 10⁶.

3. Proximity Ligation for Protein Detection

The proximity ligation procedure is analogous in certain respects to theuse of padlock probes, but is used for detecting proteins and otheranalytes. The proximity ligation procedure uses specific protein bindingagents linked to oligonucleotides. Examples of specific protein bindingagents include antibodies (defined as any specific binding agentcomprising a CDR, including phage display antibodies, single chainantibodies, monoclonal antibodies, and the like) and nucleic acidaptamers. The proximity ligation procedure is described in Landegren etal., 2003, supra; Landegren et al., 2004, Molecular tools for amolecular medicine: analyzing genes, transcripts and proteins usingpadlock and proximity probes, J Mol Recognit. 7:194-7; Gullberg et al.,2004, Cytokine detection by antibody-based proximity ligation, Proc NatlAcad Sci USA 101(22):8420-4; Fredriksson et al., 2002, Protein detectionusing proximity-dependent DNA ligation assays, Nat Biotechnol. 20:448-9;and Landegren, 2002, Methods and kits for proximity probing UnitedStates Patent Application 20020064779. Each of the these references isincorporated herein by reference. Briefly, a pair of protein bindingagents that recognize different epitopes of a target protein are used.Each of the binding agents is attached (e.g., via streptavidin-biotinlinkage) to a synthetic DNA strand that includes a PCR primer bindingsite. The synthetic DNA strands are brought into proximity when bothbinding agents bind the same target molecule. A connectoroligonucleotide that hybridizes to sequences at the ends of both of thesynthetic DNAs is added in excess, bringing termini of the DNA strandstogether so that they can be joined by ligase. In the presence of PCRreagents and primers that recognize the primer binding sites on the twoDNA strands, a region of the ligated sequence may be amplified anddetected by real time PCR. In contrast, unligated strands are notamplified and therefore not detected in the assay.

It will be appreciated that the assay also may be used to assay fornon-protein molecules that are specifically bound by a nucleic acidaptamer, antibody or other binding agent.

Proximity ligation methods can also be used to detect nucleic acidtargets. In this approach, nucleotide sequences complementary to thetarget are used rather than protein binding agents.

4. Quantitative Real Time PCR and Other Detection and QuantitationMethods

Any method of detection and/or quantitation of nucleic acids can be usedin the invention to detect a probe (PCR, SYBR Green, etc.). In oneaspect PCR (polymerase chain reaction) is used to amplify a target. Inother aspects, other amplification systems or detection systems areused, including systems described in U.S. Pat. No. 7,118,910, which isincorporated herein by reference. In other aspects, a detection systemother than PCR is used (e.g., Invader assays; PE BioSystems). In oneaspect, real time quantitiation methods are used. For example, a varietyof so-called “real time amplification” methods or “real timequantitative PCR” methods can be used to determine the quantity of atarget nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe(e.g., the “tag-specific probe”)—an approach frequently referred to inthe literature as the “TaqMan®” method. See U.S. Pat. No. 5,723,591;Heid et al., 1996, Real time quantitative PCR Genome Res. 6:986-94, eachincorporated herein by reference. It will be appreciated that while thisdisclosure often refers to “TaqMan® probes” for simplicity, theinvention is not limited to use of these probes; any tag-specific probecan be used. In particular any detection method in which thetag-specific probe is a dual-labeled fluorogenic oligonucleotide probemay be used.

5. Sample Preparation, Reagents, Related Equipment, and MolecularBiology Methods

Preparations of nucleic acids and polypeptides (“samples”) can beobtained from biological sources and prepared using conventional methodsknown in the art. PCR methods (including rt PCT) and reagents are wellknown by those of skill in the art and widely described in theliterature. Devices for thermocycling and detection of fluorescent orother signals are also known. For illustration and without limiting theinvention, examples of biological sources include samples from humansand animals (blood fractions, urine, tissue, cell extracts), plants,environmental sources (e.g., pond water) and the like.

6. Analytical Method of the Invention

It will be appreciated that preferably at least 10, more often at least25, still more often at least 50, even more often at least 100, in somecases at least 500 and sometimes at least 1000 targets are detected.Thus, the method may make use of at least 10, more often at least 25,still more often at least 50, even more often at least 100, in somecases at least 500 and sometimes at least 1000 target-specific probes.The probes may differ in the terminal sequences specific for the targetsequence, or ligands specific for an epitope, as well as at the tagsequence and/or at the primer sequences used to amplify or detect thetag.

The present invention provides new methods for detection andquantification of nucleic acids or proteins (“targets”) in one or moresamples. The methods provide:

-   -   The high specificity of the padlock probe and proximity ligation        systems.    -   The high specificity of PCR.    -   Rapid screening of >1000 nucleic acid or protein targets through        multiplexing.    -   Accurate quantitation by real-time PCR with a dynamic range at        least 3-5 orders of magnitude greater than array-based methods.

The methods can be carried out using microfluidic devices of variousconfigurations. For simplicity, the illustrative examples below describeinterrogation of 2304 targets using a Dynamic Array microfluidic devicewith a 48×48 array of reaction chambers. It will be understood thatother configurations are possible and contemplated such as, for example,48×96; 96×96; 30×120; etc.

For illustration, in the case of nucleic acid detection, the targets canbe, for example, nucleic acids associated with pathogens, RNAs for whichover- or under-expression is indicative of disease, genomic DNA, orother nucleic acids. For illustration, in the case of protein detection,targets can be polypeptides associated with pathogens, disease states,cell expression states and the like.

The assay usually has a dynamic range of at least 3 orders of magnitude,more often at least 4, even more often at least 5, even more often atleast 6, often at least 7, and sometimes at least 8 orders of magnitude.

Terminology

A Dynamic Array microfluidic device with a 48×48 matrix of reactionchambers can be thought of as having 48 columns designated #1-48 inwhich sample is introduced and 48 rows designated #1-48 in whichreagents are introduced. Positions in the array can be described by acoordinate system in which a column and row are specified. Thus [1,1](column 1, row 1), [1,48], [48,1] and [48,48] would define the four“corners” of the matrix. It will be understood that, as used in thiscontext, for illustration and not limitation, “columns” are microfluidicchannels through which sample nucleic acids or proteins are distributedto sample chambers, and “rows” are microfluidic channels through whichreagents are distributed to reagent chambers.

A “target-specific probe” means a padlock probe or a proximity probe.

A “tag detecting probe” means a probe, such as a TaqMan® probe, thatdetects and optionally quantifies a tag sequence.

“Querying” each aliquot of the sample for the presence of atarget-specific probe means determining whether a target-specific probeis associated with (e.g., bound to or cantenated with) the target, andoptionally quantifying the amount of target-specific probe in thesample.

ILLUSTRATIVE EXAMPLE 1 Analysis of Nucleic Acids from Several Samples(Specific Tags)

In this example, 48 targets are assayed in each of 48 different samples.

The 48 targets to be assayed are referred to as Targets #1-48. For eachtarget a padlock oligonucleotide probe is designed having terminispecific to the target (referred to by number), universal PCR primersites, and one of 48 tag sequences. Thus, for target #4, the padlockoligonucleotide probe is padlock probe #4 and the tag sequence is one oftags #1-48.

For illustration, the assay may be carried out as follows:

-   -   1) Process each sample by        -   a) adding the 48 padlock oligonucleotide probes;        -   b) ligating those oligonucleotides that hybridize to a            target molecule, thereby producing circular cantenated            probes; and        -   c) adding exonuclease to remove any unhybridized probes.    -   In the resulting processed sample, from 0 (if none of the        targets is present in the sample) to 48 (if all of the targets        are present in the sample) versions of circularized padlock        oligonucleotide probes are present, each characterized by a        different tag.    -   2) Load Sample 1 into the column 1 sample channel and distribute        to 48 sample chambers. Load Samples 2-48 into columns 2-48,        respectively, and distribute each sample to 48 sample chambers.        A total of 2304 chambers are loaded.    -   3) Via the Row 1 reagent channel, distribute universal primers,        PCR reagents and a TaqMan® probe that recognizes Tag #1 to the        reagent chambers in row 1. Via the Row 2-48 reagent channels,        distribute universal primers, PCR reagents and TaqMan® probes        that recognize Tags #2-48, respectively, to the corresponding        rows.    -   4) Carry out real-time PCR and monitor.    -   5) Analysis:    -   Readout for [1, 1] is for target 1 in Sample 1.    -   Readout for [1, 2] is for target 2 in Sample 1.    -   Readout for [2, 3] is for target 3 in Sample 2.    -   Etc.        Also see FIG. 2A.

ILLUSTRATIVE EXAMPLE 2 Analysis of Nucleic Acids in One Sample (SpecificTags)

In this example, 2304 targets are assayed in a single sample.

The targets to be assayed are referred to as Targets #1-2304. For eachtarget a padlock oligonucleotide probe (2304 probes total) is designedhaving (i) termini specific to the target, (ii) universal PCR primersites, and (iii) one of 48 tag sequences. Thus, for target #4 thepadlock oligonucleotide probe is padlock probe #4 and the tag sequenceis one of tags #1-48. More specifically,

Target Padlock probe # Tag sequence  1-48  1-48 1-48, respectively 49-9649-96 1-48, respectively  97-146  97-146 1-48, respectively . . . . . .. . . 2255-2304 2255-2304 1-48, respectivelyThe assay is carried out as follows:

-   -   1) Divide the sample into 48 aliquots.    -   2) Process each aliquot by        -   a) adding 48 padlock oligonucleotide probes to each aliquot            (probes 1-48 to Sample 1, probes 49-96 to Sample 2, probes            97-146 to Sample 3, etc.);        -   b) ligating those oligonucleotides that hybridize to a            target molecule thereby circularizing them; and        -   c) adding exonuclease to remove any unhybridized probes.    -   In the resulting processed aliquots (combined), from 0 (if none        of the targets is present in the sample) to 2304 (if all of the        targets are present in the sample) versions of circularized        padlock oligonucleotide probes are present, each characterized        by one of tags 1-48.    -   3) Load aliquot 1 into column 1, and aliquots 2-48 into columns        2-48, respectively, and distribute to 2304 sample chambers.    -   4) Via the Row 1 reagent channel, distribute universal primers,        PCR reagents and a TaqMan® probe that recognizes Tag #1 to the        reagent chambers in row 1. Via the Row 2-48 reagent channels,        distribute universal primers, PCR reagents and a TaqMan® probe        that recognizes Tags #2-48, respectively.    -   5) Carry out real-time PCR and monitor.    -   6) Analysis        -   Readout for [1, 1] is for target 1.        -   Readout for [1, 2] is for target 2.        -   Readout for [2, 3] is for target 51.        -   Readout for [48, 48] is for target 2304.        -   Etc.

ILLUSTRATIVE EXAMPLE 3 Analysis of Proteins

This example describes an assay of each of 48 proteins in 48 differentsamples.

The 48 proteins to be assayed are referred to as Proteins #1-48. Foreach protein a pair of monoclonal antibodies that binds the protein, atdifferent epitopes, is obtained. They are referred to as antibody pairs#1-48. Each antibody is attached to a synthetic DNA strand. Eachantibody pair has a unique set of two stands such that each pair ofstrands is recognized by a unique selector oligonucleotide (selectoroligonucleotides #1-48). Moreover, the DNA strand sequences are selectedso that when any two strands are joined, the resulting sequence includesone of predetermined tag sequences (Tags #1-48) flanked by two PCRuniversal primer binding sites. Prior to ligation, each synthetic DNAstrand has only one of the two PCR binding sites necessary for PCRamplification (i.e., PCR amplification is possible only from the ligatedmolecules).

The assay is carried out as follows:

-   -   1) Process each sample by        -   a) adding antibody pairs #1-48;        -   b) adding selector oligonucleotides #1-48;        -   c) ligating those DNA strands that are joined by the            selector oligonucleotide, based on the proximity of the            antibodies to which the DNA strands are attached;    -   In the resulting processed sample, from 0 (if none of the        targets is present in the sample) to 48 (if all of the targets        are present in the sample) versions of ligated DNAs will be        present, each characterized by a different tag.    -   2) Load Sample 1 into the column 1 sample channel and distribute        to 48 sample chambers. Load Samples 2-48 into columns 2-48,        respectively, and distribute each sample to 48 sample chambers.        A total of 2304 chambers are loaded.    -   3) Via the Row 1 reagent channel, distribute universal primers,        PCR reagents and a TaqMan® probe that recognizes Tag #1 to the        reagent chambers in row 1. Via the Row 2-48 reagent channels,        distribute universal primers, PCR reagents and TaqMan® probes        that recognizes Tags #2-48, respectively to the corresponding        rows.    -   4) Carry out real-time PCR and monitor.    -   5) Analysis:    -   Readout for [1, 1] is for target 1 in Sample 1.    -   Readout for [1, 2] is for target 2 in Sample 1.    -   Readout for [2, 3] is for target 3 in Sample 2.    -   Etc.

In Examples 1-3, the PCR amplification step uses universal PCR primerswith corresponding universal PCR primer sites on the padlock orproximity probes. Specificity in these methods is due in part to thespecificity of the padlock/proximity probe to the target and thespecificity of the TaqMan® probes for specific padlock/proximitysequences. In an alternative embodiment, a “universal” TaqMan® probe isused, but the PCR primer sites on the padlock/proximity probes vary. Inthese embodiments, specificity is due in part to the specificity of thepadlock/proximity probe to the target and the specificity of the PCRprimers to specific padlock/proximity sequences. In certain applicationsthis approach has advantages. For example, there may be considerablecost savings in using one TaqMan®-type probe, rather than many. Inaddition in the method of Example 1, supra, the universal PCR primers inany reaction chamber may amplify several different amplicons (ifmultiple target sequences are present in the chamber). This may reducespecificity, especially in cases in which one target is present inexcess over the target corresponding to the specific TaqMan® probeintroduced into the chamber.

Although introducing specificity using specific PCR primers and specificTaqMan® probes (as well as the specificity of the padlock/proximityprobe for the target) has been described herein, it will be understoodby those of skill that intermediate and additive combinations of theseapproaches may also be used.

ILLUSTRATIVE EXAMPLE 4 Analysis of Nucleic Acids in 48 Samples (SpecificPCR Primers)

This Example 4 is analogous to Example 1, in that 48 samples areassayed. Also see FIG. 2B. In this example, 48 targets are assayed ineach of 48 different samples.

The 48 targets to be assayed are referred to as Targets #1-48. For eachtarget a padlock oligonucleotide probe is designed having terminispecific to the target (referred to by number), a universal tagsequence, and one of 48 pairs of PCR primer binding sequences flankingthe tag (bound by one of Primer Pairs #1-48). The assay is carried outas follows:

-   -   1) Process each sample by        -   a) adding the 48 padlock oligonucleotide probes;        -   b) ligating those oligonucleotides that hybridize to a            target molecule, thereby producing circular cantenated            probes; and        -   c) adding exonuclease to remove any unhybridized probes.    -   In the resulting processed sample, from 0 (if none of the        targets is present in the sample) to 48 (if all of the targets        are present in the sample) versions of circularized padlock        oligonucleotide probes are present, each characterized by a        different tag.    -   2) Load Sample 1 into the column 1 sample channel and distribute        to 48 sample chambers. Load Samples 2-48 into columns 2-48,        respectively, and distribute each sample to 48 sample chambers.        A total of 2304 chambers are loaded.    -   3) Via the Row 1 reagent channel, distribute the universal        TaqMan® probe, PCR reagents and one PCR primer pair. The primer        pair that recognizes padlock probe #1 is added to the reagent        chambers in row 1. Via the Row 2-48 reagent channels, distribute        primer pairs that recognize padlock probes #2-48, are added.    -   4) Carry out real-time PCR and monitor.    -   5) Analysis:    -   Readout for [1, 1] is for target 1 in Sample 1.    -   Readout for [1, 2] is for target 2 in Sample 1.    -   Readout for [2, 3] is for target 3 in Sample 2.    -   Etc.

ILLUSTRATIVE EXAMPLE 5 Analysis of Nucleic Acids in One Sample (SpecificPCR Primers)

In this example, 2304 targets are assayed in a single sample. Also seeFIG. 3.

The targets to be assayed are referred to as Targets #1-2304. For eachtarget a padlock oligonucleotide probe (2304 probes total) is designedhaving (i) termini specific to the target, (ii) a universal tag sequenceand (iii) a unique PCR primer site pair (referred to as forward primersF_(N) and reverse primers R_(N), where N is 1 to 48). Thus, for target#4 the padlock oligonucleotide probe is padlock probe #4, and the PCRprimer pair is defined by one of forward primers #1-48 and one ofreverse primers #1-48.

The assay is carried out as follows:

-   -   1) The padlock (or in alternative embodiments, proximity) assay        is carried out using 2304 padlock probes;    -   2) The Sample is divided into 48 aliquots and forward primers        are added as follows:

Forward Primer Target Padlock probe # sequence  1-48  1-48 1-48,respectively 49-96 49-96 1-48, respectively  97-146  97-146 1-48,respectively . . . . . . . . . 2255-2304 2255-2304 1-48, respectively

-   -   3) Load aliquot 1 into column 1, and aliquots 2-48 into columns        2-48, respectively, and distribute to 2304 sample chambers.    -   4) Via the Row 1 reagent channel, distribute PCR reagents, the        universal TaqMan® probe, and the reverse primer (e.g., R₁)        corresponding to Target 1. Via the Row 2-48 reagent channels,        distribute PCR reagents, the universal TaqMan® probe, and one of        the reverse primers 2-48 (e.g., R₂₋₄₈), respectively.    -   5) Carry out real-time PCR and monitor.    -   6) Analysis        -   Readout for [1, 1] is for target 1.        -   Readout for [1, 2] is for target 2.        -   Readout for [2, 3] is for target 51.        -   Readout for [48, 48] is for target 2305.        -   Etc.            It will be appreciated by one guided by this disclosure that            that numerous variations of methods described in these            examples may be carried out, and that the examples above are            for illustration and not intended to limit the invention in            any fashion. It will further be appreciated that, although            the examples above describe a device with a 48×48 matrix of            reaction chambers and analysis of one or 48 samples are            analyzed, many other combinations are possible. For example,            many other matrices may be used (as described above) and            intermediate numbers of samples may be analyzed. For            example, in a 48×48 matrix the number of samples may be any            divisor of 48 that gives a whole integer product. More            generally, a C×R array can be used to assay the number n            samples for CR/n targets, where C/n is a whole integer.

EXAMPLE 6 Dynamic Array Device

As noted above, one example of a dynamic array device that can be usedto practice the methods of the invention is the “48.48 Dynamic Array”(Fluidigm Corp., S. San Francisco Calif.). FIG. 1 depicts the 48.48Dynamic Array with an integrated input frame.

The 48.48 Dynamic Array provides experiment throughput six-fold higherthan a 384-microwell plate, while delivering quantitative data on a parwith real-time qPCR standards. This nanofluidic chip for TaqMan® assaysalso provides substantial reductions in time and running costs tocomplete large-scale gene-expression studies. With the 48.48 chip,laboratories realize the following advantages:

-   -   Flexibility to assay any 48 samples against any 48 genes.    -   Throughput of 2,304 data points per run equivalent to all        pair-wise combinations (48×48) of samples and assays loaded into        a chip.    -   Parallel multiplexing of 48 genes per sample.    -   Only ninety-six liquid-transfer steps for setup.    -   Reaction volumes of less than ten nanoliters.    -   Reliable single-copy qPCR detection within reaction chambers.        Higher Throughput, Streamlined Operations

The 48.48 Dynamic Array provides throughput far beyond what is possiblewith other technologies, yet reduces complexity. The chip's input framehas a footprint and spacing of fluid microwells that are SBS-compliant.For this reason, standard equipment may be used to handle the transferof samples and TaqMan® assays. The chip's heat spreader, a flat siliconlayer bonded to the underside of each chip, ensures good thermal contactnecessary for uniform and accurate temperatures among all the PCRreactions. In addition to dynamic arrays, Fluidigm provides hardware andsoftware to automate the entire setup. After assay components have beentransferred to the input frame, the NanoFlex™ IFC Controllerautomatically drives the fluids to respective reaction chambers. TheBioMark™ System performs thermal cycling and real-time detection andanalysis. Each chip has a unique barcode, which is scanned into thesystem's computer for ease of tracking

Efficient Multiplexed Readouts

Dynamic arrays allow unlimited flexibility in experiment setup as wellas multiple data points per sample. After any 48 samples and any 48assays have been transferred to the input frame, the chip's internalnetwork does the work of routing and combining them into 2,304 (48×48)reactions forty-eight readouts for each of 48 samples. Because dynamicarrays precisely partition samples and assays into 10 nanoliterreactions, it is unnecessary to use endogenous controls in each reactionto account for variations in sample volumes. If desired, one or more ofthe 48 assays may serve as endogenous controls if they are for genesknown to be consistently expressed from sample to sample.

Smoother Workflow, Faster Answers

Relative efficiency may also be viewed in light of the density ofexperiments per run. Managing a gene expression study involving 2,000samples against a set of 48 genes would require two-hundred-fifty384-microwell plates as compared to forty-two 48.48 Dynamic Arrays.Completing such a study would take 25 days using microplates compared tofour and a half days using dynamic arrays. This increased throughput isachieved with only 96 liquid-transfer steps per chip compared to the4,608 steps for equivalent throughput on microplates. The radicalreduction saves time but also reduces the cost of pipette tips andminimizes errors, sample loss, and freeze-thawing of reaction componentsassociated with microplate-based operations. Taken together, theadvantages of dynamic arrays elevate real-time qPCR to an efficienttechnique for high-throughput gene expression studies.

Dynamic Array Specifications The 48.48 Dynamic Array for gene expressionanalysis is a nanofluidic chip for real-time quantification of PCRproducts. Components Specifications Sensitivity Single copies can beroutinely detected if present in the reaction chamber Sample inputs 48TaqMan assay inputs 48 Minimum sample/assay input  5 microliters volume(per frame used) Reactions per chip  2.304 Number of liquid transfer 96steps to load chip Volume per reaction 10 microliters Sample and TaqManassay Unique barcode on each chip tracking

* * *

Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims which follow. Allpublications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same. Theinvention having now been described by way of written description andexample, those of skill in the art will recognize that the invention canbe practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

The invention claimed is:
 1. An assay method for detection of aplurality of nucleic acid or protein targets in a sample comprising (a)combining target-specific probes with the sample under conditions inwhich at least 25 target-specific probes each bind a different specifictarget in the sample, if the specific target is present, wherein eachsaid target-specific probe comprises a common nucleic acid tag sequenceand a unique combination of a first primer binding site and a secondprimer binding site flanking the nucleic acid tag sequence, wherein atleast two of said target-specific probes do not have the same firstprimer binding sites and at least two of said target-specific probes donot have the same second primer binding sites, and wherein thetarget-specific probe forms a target-specific probe product associatedwith the target if the target is present in the sample, therebyassociating the tag with the target by forming a target-specific probeproduct associated with the target; (b) segregating aliquots of thesample; (c) querying each said aliquot for the presence or absence of adifferent target-specific probe product, wherein said querying comprisesamplifying the nucleic acid tag sequence using a first primer that bindsthe first primer binding site and a second primer that binds the secondprimer binding site; and detecting the presence or absence of the commonnucleic acid tag sequence using a tag-detecting probe.
 2. The method ofclaim 1 wherein target-specific probes sufficient to identify at least500 different targets are combined with the sample.
 3. The method ofclaim 1 wherein said querying comprises quantifying the amount of thenucleic acid tag sequence in each aliquot and correlating the amount ofthe tag sequence with the amount of target in the sample.
 4. The methodof claim 3 in which the tag sequence is amplified using PCR.
 5. Themethod of claim 4 wherein the amplified tag sequence is detected using afluorogenic nuclease assay.
 6. The method of claim 5 wherein theamplified tag sequence is quantified by the continuous measurement ofPCR product accumulation using a dual-labeled fluorogenicoligonucleotide probe.
 7. The method of claim 1 wherein the target is anucleic acid molecule.
 8. The method of claim 7 wherein thetarget-specific probe is a padlock probe.
 9. The method of claim 1wherein the target is a protein molecule.
 10. The method of claim 9wherein the target-specific probe is a proximity probe.
 11. The methodof claim 1 wherein the assay has a dynamic range of at least 4 orders ofmagnitude.
 12. The method of claim 1 wherein querying each aliquot ofthe sample for the presence of a different target-specific probecomprises combining each aliquot with a composition containing PCRprimers and/or a tag detecting probe.
 13. The method of claim 12 whereinquerying each aliquot of the sample for the presence of a differenttarget-specific probe comprises combining each aliquot with acomposition containing PCR primers and a tag detecting probe.
 14. Themethod of claim 13 wherein the tag-detecting probe is a dual-labeledfluorogenic oligonucleotide probe.
 15. The method of claim 14 whereineach aliquot is queried using the same tag-detecting probe and adifferent PCR primer pair.
 16. The method of claim 14 wherein eachaliquot is queried using the same PCR primer pair and a differenttag-detecting probe.
 17. An assay method for detection of a plurality ofDNA targets in a sample comprising (a) combining target-specific padlockprobes sufficient to identify at least 25 different targets with thesample under conditions in which the probes bind and cantenate to thetarget molecules, if present, forming a target-specific probe product,wherein each probe comprises a common nucleic acid tag sequence and aunique combination of a first primer binding site and a second primerbinding site, wherein at least two of said target-specific probes do nothave the same first primer binding sites and at least two of saidtarget-specific probes do not have the same second primer binding sites,and wherein the padlock probes form target-specific probe productsassociated with the target if the target is present in the sample,thereby associating the tag sequence with the target; (b) segregatingaliquots of the sample; (c) querying each aliquot for the presence of adifferent probe molecule catenated to the target, wherein said queryingcomprises detecting the tag sequence if present in the aliquot, andwherein detecting the tag comprises amplifying the tag sequence using afirst primer that binds the first primer binding site and a secondprimer that binds the second primer binding site and detecting theamplification using a dual-labeled fluorogenic oligonucleotide probe.